RESULTS AND DISCUSSION

ERDC TN-DOER-C30

February 2003

Hygromycin to select for cells carrying the pIND plasmid. Colonies were again generated from

single cells, and screened to isolate the one with the highest activity, which was then designated

HepG2-EcR.

HepG2-EcR Assay. For details, see Appendix I. Briefly, cells were plated in 96-well plates at

40K cells per well. After the cells were allowed to attach overnight, cells were treated with A1242

(1, 10, 100 and 500 ppb) in the presence of 0, 1, 3, 10, 30, or 100 M Ponasterone A. That is, six

series of dose combinations were prepared in which each of the A1242 concentrations was combined

with one of the Ponasterone A mass concentrations. Additionally, one set of cells was treated with

Ponasterone A alone at the five 1-100 M mass concentrations. After 48 hr, cells were lysed

in 100 L of lysis buffer and the lysate analyzed for -galactosidase activity.

-Galactosidase Assay. The lysate (50 l) was transferred to a 96-well plate, and Buffer A

added to each sample (110 L; 100 mM NaH2PO4, pH 7.5, 10 mM KCl, 1 mM MgSO4, 50 mM

-mercaptoethanol). After a 5-min pre-incubation at 37 C, 50 L of substrate was added

(4 mg/mL of o-nitrophenyl-p-galactopyranoside (ONPG) to each well. Immediately after addition

of the substrate, the absorbance was measured at 405-420 nm every minute for 15 min in a

temperature-controlled (37 C) plate reader.

Data Analysis. Data from five separate experimental runs were combined by normalizing the

activity to the maximal activity achieved with Ponasterone A alone. Data were plotted as log-dose

versus response to determine whether there were shifts in the effective doses for either 50 percent

maximal activity (EC50), or for maximal activity (ECmax) in the presence of A1242. Data from

10- M Ponasterone A co-exposures were compared to 10 M Ponasterone A to determine whether

this single endpoint is a viable alternative to running complete dose response curves.

Crawfish Molting Assay. In separate experiments, juvenile crawfish (<1.5 cm) were held

individually in 22-mL vials, with water changes twice daily. They were fed once daily with crushed

catfish food pellets. Animals were held until molting was observed, at which time exposures were

begun. Exposures included nondosed controls, solvent controls (DMSO), and 100, 500, or 1,250

ppb A1242; exposures continued until a second molting was observed.

RESULTS AND DISCUSSION

HepG2-EcR Assay. Enzymatic activity as changes in optical density (mOD/min) at five

Ponasterone A mass concentrations (1, 3, 10, 30, and 100 M) is shown in Figure 2. The data are

normalized to the activity of 100 M Ponasterone A alone (maximal induced activity). No

significant changes were observed in the effective dose for EC50 in the cells, but a depression in the

ECmax activity was observed with the 500-ppb A1242 dose in the presence of 10-100 M

Ponasterone A, indicating that A1242 disrupts the normal ligand-receptor interaction. In an effort

to streamline the testing protocols, the A1242 dilutions in the 10- M Ponasterone A doses were

compared to determine whether responses at that single dose are indicative of responses observed

in the full dose-response curve. Activity was significantly suppressed in both the 500- and 100-ppb

A1242 exposures when compared to Ponasterone A alone (Figure 3), and the suppression appeared

to be dose-related (r2 = 0.80 for log-dose versus response regression).